The invention relates to a novel use of a Bordetella adenylcyclase toxin in the manufacturing of vectors for targeting in vivo a molecule of interest, specifically to CD11b expressing cells. The invention also relates to an immunogenic composition that primes immune responses, to pharmaceutical compositions and a new vector for molecule delivery to CD11b expressing cells.
Bordetella pertussis, the causative agent of whooping cough, secretes several toxins including the well-known pertussis toxin (PT) and the adenylate cyclase toxin (CyaA) or also adenylcyclase. CyaA is a critical virulence factor of B. pertussis in the murine respiratory model that is required for the early steps of lung colonization. Indeed, genetic deletion of this toxin dramatically decreases the pathological effects of B. pertussis infection, reducing the number of bacteria recovered from the lung and almost abolishing the inflammatory cell recruitment and the lung lesions observed after infection [Weiss et al., 1984; Weiss et al., 1989; Gross et al., 1992; Khelef et al., 1992; Khelef et al., 1994; Gueirard et al., 1998]. Moreover, CyaA is an antigen protective against B. pertussis infection in the murine respiratory model [Guiso et al., 1989; Guiso et al., 1991].
Originally discovered by Hewlett et al in B. pertussis culture supernatants [Hewlett et al., 1976], the adenylcyclase was later found to be activated by the eukaryotic calmodulin [Wolff et al., 1980]. This striking feature quickly found a rationale when it was shown by Confer and Eaton that the adenylcyclase could enter into eukaryotic cells where, upon activation by calmodulin, it could trigger a large increase in cAMP within these target cells [Confer et al., 1982].
Adenylcyclase is encoded by the cyaA gene, and its expression, like that of other virulence genes of B. pertussis, is coordinately regulated by environmental signals. The cyaA gene is part of an operon that also contains genes cya B, D and E, that are required for CyaA secretion [Ladant et al., 1999].
The CyaA toxin is a bifunctional protein of 1706 residues that is made of a N-terminal catalytic domain of 400 amino acids and a C-terminal part of 1306 residues which is responsible for the binding of the toxin to target cell membrane and the subsequent delivery of the catalytic moiety into the cell cytosol [Sakamoto et al., 1992] [Ladant et al., 1999]. This part also exhibits a weak hemolytic activity due to its ability to form cation-selective channels in biological membranes [Benz et al., 1994] [Gray et al., 1998]. This region is homologous to Escherichia coli hemolysin and other members of the RTX (Repeat in ToXin) family of bacterial toxins. In particular, it contains a series of glycine and aspartate-rich nonapeptide repeats that are involved in calcium binding [Rose et al., 1995] [Coote et al., 1992].
The CyaA polypeptide is synthesized as an inactive protoxin that is converted to an active toxin by posttranslational palmitoylation of two internal lysines (lysines 856 and 963). This modification requires the product of an accessory gene, cyaC, which is located nearby cyaA on B. pertussis chromosome.
CyaA has been shown to bind to and invade a variety of cell types including cells lacking membrane traffic like mammalian erythrocytes [Rogel et al., 1992]. This suggested that the catalytic domain of CyaA is directly translocated across the plasma membrane of target cells. The internalization of the catalytic domain into the cell cytosol is calcium and temperature-dependent and depends upon the plasma membrane potential [Rogel et al., 1992] [Karimova et al., 1998] [Otero et al., 1995]. However, the molecular mechanisms by which the toxin transports its N-terminus catalytic domain across the membrane remain largely unknown to date. Furthermore no specific receptor has been reported for CyaA binding.
The physiological consequences of cellular intoxication by CyaA were characterized in vitro in phagocytes. Confer and Eaton first showed that the Cya A extracted from B. pertussis increases the intracellular cAMP level in neutrophils or macrophages leading to an inhibition of chemotaxis and bactericidal functions such as superoxide generation and phagocytic abilities [Confer et al., 1982]. These activities were later confirmed with purified toxins or with bacterial mutants genetically deleted of CyaA [Pearson et al., 1987; Friedman et al., 1987] [Njamkepo at al., 2000]. On the contrary, and despite significant changes in their cAMP content, the viability of cell lines from non-hematopoietic origin appeared to be unaffected by the CyaA intoxication [Bassinet et al., 2000]. Moreover, the present inventors previously demonstrated that B. pertussis CyaA can trigger macrophage apoptosis in vitro [Khelef et al., 1993; Khelef et al., 1995] and in vivo [Gueirard at al., 1998]. In these models, genetic deletion of CyaA abolished macrophage apoptosis, but not neutrophil death, suggesting that CyaA i) is mainly responsible for macrophage apoptosis, ii) might be responsible for neutrophil apoptosis, but that another factor may also be responsible.
Besides that, in vivo studies performed in a murine model of B. bronchiseptica infection (the animal homologue of B. pertussis whose CyaA is closely related) demonstrated that the major target of B. bronchiseptica CyaA toxicity is a GM-CSF-dependent and cyclophosphamide-sensitive population that controls the early steps of infection [Harvill et al., 1999]. These criterions identified neutrophils and possibly other cells including macrophages or dendritic cells but no data is disclosed or suggested that the CD11b cell receptor is involved in the targeting by Cya A. These populations of target cells for CyaA is the same that limits the early phases of infection and favors the development of an adaptive immune response that controls the latter phases of infection [Harvill et al., 1999].
Unlike other toxins, CyaA has been considered for a long time, as independent of any receptor binding. This is based on the observations that i) CyaA can intoxicate a wide variety of model cell lines from various origin [Ladant et al., 1999] CyaA binds to Jurkat cells and sheep erythrocytes in a non saturable fashion [Gray et al., 1999]. However, some specificity has been found in respect of cells infected by CyaA. Indeed, in vivo studies showed that during murine respiratory infection with Bordetella species, CyaA destroyed specifically leukocytes (especially macrophages) without damaging dramatically epithelial cells [Gueirard et al., 1998; Harvill et al., 1999].
It has been proposed in patent application WO 93/21324 to use the recombinant Bordetella adenylcyclase to induce a CD4+ T cell or a CD8+ T cell response; however, since no specific receptor for Bordetella adenylcyclase was identified, it was not known if the antigen presentation was related to uptake by non professional antigen presenting cell followed by cross-priming and presentation by dendritic cells or if the antigen was targeted to professional Antigen Presenting Cells (pAPC).
In line with their surface phenotype, dendritic cells (high expression of MHCl and II, costimulatory and adhesion molecules) represent the most potent APC in many in vitro assay for the priming of naive T cells [Bell et al., 1999; Viola et al., 1999]. Other APC like resting naive B cells, for example, could even be tolerogenic since injection of resting, male B cells into female hosts leads to the specific tolerization of male-specific CD8+ T cells [Fuchs et al., 1992]. In vitro, naive B cells could delete naive CD8+ T cells via a Fas dependent-mechanism [Bennett et al., 1998].
Moreover, Ag presentation by dendritic cells correlates in vivo with the induction of T cell responses. This has been established for MHCII-restricted Ag presentation. Adjuvant-free Ag injection via the intravenous (iv) route usually does not induce T cell priming [Kyburz et al., 1993; Aichele et al., 1994; Aichele et al., 1995] and leads to Ag presentation by non-specific B cells [Guery et al., 1997] [Zhong at al., 1997; Reis e Sousa et al., 1999] and, eventually dendritic cells [Crowley et al., 1990] [Zhong et al., 1997; Reis e Sousa et al., 1999]. In contrast, local immunization strategies like subcutaneous (sc) immunization in the presence of adjuvant usually, induces T cell priming and targeted Ag presentation by Langerhans cell migrating from the skin to the LN draining the immunization site. In this case, B cells and macrophages are not involved [Guery et al., 1996]. Similar results were obtained after sc or intradermic (id) DNA immunization for MHCII and MHCl-peptide complexes: dendritic cells can be directly transfected at the local site of injection and then migrate to the afferent LN via afferent lymphatics [Condon at al., 1996; Casares et al., 1997; Porgador at al., 1998]. The migration is known as a key event of immunity since mechanical disruption of afferent lymphatics abrogates T cell response to skin sensitizers or skin grafts [Zinkemagel et al., 1997].
Therefore, targeting to dendritic cells is essential for CD4+ and CD8+ T cell stimulation. Since most antibody responses are dependent upon CD4+ T cell help, targeting antigen to dendritic cells is a major goal in vaccination.
Applicants have been interested in studying the presentation of adenylcyclase of Bordetella species by T cell, and have identified a specific receptor molecule present on specific cells, that interacts with CyaA and opens new possibilities for the use of CyaA as a proteinaceous vector for molecules of interest.
Genetically detoxified bacterial toxins represent candidates as vaccine vectors, in particular for T epitope, due to their ability to invade eukaryotic cells (Ladant et al., 1999). However, few proteinaceous vectors were shown to prime CTL responses in vivo (Ballard et al., 1996, Cabonette et al., 1999). Moreover, despite numerous in vitro promising studies, no vector was shown to be exclusively targeted to pAPC particularly to dendritic cells and more particularly to myeloid dendritic cells.
The inventors have further shown that other cells, especially neutrophils, could be targeted by the vectors of the present invention.
The invention provides means that may at least in part, fulfil these needs and proposes new vectors that would specifically target molecules to determined populations of pAPC for example, to enable stimulating immune response.
Moreover, molecule targeted to the pAPC and specific leukocytes would enable the manufacturing of new vectors useful to deliver biologically active molecule to the proximal environment of these cells. These molecules, for example, could modulate the functional properties of the targeted cells or those involved in the immune response or in the inflammatory response.
Indeed, the inventors have found that Bordetella pertussis adenylcyclase toxin binds specifically with a cellular receptor designated (CD11b/CD18) αM/β2 receptor and that this interaction is required for the intracellular delivery of the adenylcyclase domain to the cytosol of cells and subsequent for cell death. αM/β2 (CD11b/CD18) integrin is a dimer of the β2 integrin family, the expression of these integrins being restricted to leukocytes. CD11b/CD18 αM/β2 displays a pattern of expression in mouse and human, which is restricted to neutrophils/granulocytes, macrophages, dendritic cells, NK cells and subsets of B and T CD8+ lymphocytes (Jeyaseelan et al., 2000, Arnaout et al., 1990).
Therefore, this receptor would represent an ideal target for new vectors, designed in particular for T epitope immunization.
Applicants have shown in the present invention that the Bordetella adenylcyclase can be used to target a molecule in vivo specifically to CD11b expressing cells.
In particular, Applicants have shown in the present invention that a peptide antigen comprised in the Bordetella pertussis adenylcyclase toxin can efficiently be targeted specifically to the surface of dendritic cells, translocated in the cytosol of said dendritic cells and prime a CTL response.
In a specific embodiment, said response is obtained bypassing adjuvant requirement and CD4+ T-cell help.
It has also been shown that genetically modified adenylcylase can be chemically coupled to a peptide of interest to target said peptide to CD11b expressing cells, especially the cytosol of dendritic cells.
This invention thus provides new efficient immunogenic composition as well as new drug delivery vector to CD11b expressing cells.